Programmed death-ligand 1 expression and overall survival in Thai patients with gastric cancer

Programmed death-ligand 1 (PD-L1) expression has now been implicated in gastric cancer (GC). This study was conducted to determine the impact of clinicopathological characteristics on PD-L1 expression and its association with survival in GC patients receiving standard-of-care. In total, 268 GC patients receiving upfront surgery were enrolled at Chiang Mai University Hospital. PD-L1 expression was assayed by immunohistochemistry staining using the Dako 22C3 pharmDx. The rates of PD-L1 positivity by combined positive score (CPS) at a cutoff value of 1 and 5 were 22% and 7%. PD-L1 positivity was significantly higher in patients younger than 55 than those older than 55 (32.6% vs. 16.5%, p = 0.003; 11.6% vs. 4.4%, p = 0.027). PD-L1 positivity was observed more frequently in GC with metastases than without (25.2% vs. 17.1%, p = 0.112; 7.2% vs. 6.7%, p = 0.673). Patients with PD-L1 positive had a significantly shorter median overall survival than those with PD-L1 negative (32.7 vs. 41.6 months, p = 0.042, 27.6 vs. 40.8 months, p = 0.038). In conclusion, PD-L1 expression has been associated with young age, short survival, and metastases, although unrelated to the tumor stage. For GC patients, PD-L1 testing is recommended, especially among young patients with metastases.


Immunohistochemistry (IHC) staining and evaluation. IHC was performed on 4 -µm-thick tissue
sections using an automated IHC Stainer (Ventana, Tucson, AZ, USA). The assessment of PD-L1 protein expression in GC is a qualitative IHC assay that uses anti-PD-L1 antibodies (Dako, 22C3) to detect PD-L1 protein in formalin-fixed, paraffin-embedded tissues from gastric adenocarcinomas. A minimum of 100 tumor cells must be present in the PD-L1 stained slide for the specimen to be considered adequate for PD-L1 evaluation. Expression of PD-L1 was reported as CPS, defined as the total number of PD-L1 positive cells (lymphocytes, macrophages, and tumor cells) divided by the total number of viable tumor cells 10 . The CPS ≥ 1 and ≥ 5 were chosen to define PD-L1 positive. A monoclonal antibody against Latent Membrane Protein (LMP)-1 (CS1-4; Dako, Glostrup, Denmark) was used to detect EBV-specific protein to identify EBV status for GC with CPS ≥ 1. IHC for LMP-1 was done according to the method previously described 11 . Brown granular cytoplasmic and membrane staining was interpreted as positive for EBV LMP-1, whereas bluish staining of the cytoplasm and membrane was interpreted as negative for EBV LMP-1. A positive control included a tissue known to have EBV infection, whereas, for negative controls, the test antibody was omitted and replaced by phosphate-buffered saline.
Statistical analysis. All statistical analyses were performed using Stata software, version 15.1 MP (Stata Corporation, College Station, Texas, USA). Data for categorical variables was shown by frequency and percentage. As appropriate, the comparison between PD-L1 expression and clinicopathological features of GC was analyzed using the Chi-squared test or Fisher's exact test. Overall survival was defined as the time from the initial diagnosis to death by any cause or last follow-up. The relationship between PD-L1 expression and overall survival was analyzed using the Kaplan-Meier method and log-rank tests, with PD-L1 negative as the reference. A two-tailed p value < 0.05 was considered statistically significant.

Consent to participate.
Patients received written and oral information on the study and gave their consent to participate and use their medical data for research purposes.

Results
Clinicopathological characteristics. A total of 268 patients with GC were included in this study. None of the patients received chemotherapy or radiation before surgery. There were 132 (49%) males and 136 (51%) females with a mean age of 59.0 ± 10.2 years (range 37-87 years) at diagnosis. Tumor location was in the lower part of the stomach for 51%, the middle part for 28%, and the upper part for 21%. A tumor diameter of less than 5 cm accounted for 49% of patients, whereas a tumor diameter of more than 5 cm accounted for 51%. Lauren classification was diffuse for 55% and intestinal type for 45%. According to the pTNM classification, the disease was stage I, II, III, IV, and undetermined at 3%, 12%, 25%, 52%, and 8%, respectively. Lymph node metastasis was 44%, and vascular invasion was 23%. More detailed clinicopathological characteristics are summarized in Table 1.

Expression of PD-L1 and clinical outcomes.
We investigated the prognostic significance of PD-L1 expression concerning overall survival. Based on CPS, overall survival is represented by the Kaplan-Meier curve in Figs. 1 and 2. Our study revealed that the median overall survival was significantly shorter in patients with

Discussion
PD-1/PD-L1 immune checkpoint inhibitors are now approved for treating patients with advanced GC 12,13 . PD-L1 expression, evaluated by IHC, is accepted as a predictive biomarker for the effectiveness of PD-1/PD-L1 inhibitors 14 . This present study is the first evaluation of PD-L1 expression in Thai patients with GC. The prevalence of PD-L1 expression with a CPS cutoff value of 1 and 5 in patients with GC was 22% and 7%, respectively. Patients with PD-L1 positive were typically younger and had significantly shorter survival than those with PD-L1 negative. PD-L1 expression is common in GC patients with metastases. PD-L1 overexpression appears to be an unfavorable prognostic factor in GC.
Our study describes findings from the clinical audit of PD-L1 expression in GC, providing the first insight into the rate of PD-L1 positivity in gastric adenocarcinoma in Thailand. Based on 268 cases of GC analyzed for PD-L1 expression, patients with PD-L1, CPS ≥ 1, and PD-L1, CPS ≥ 5 accounted for 22% and 7% of participants, respectively. The rate of PD-L1 positivity was lower than that reported in the literature from different populations (43% to 63%) [15][16][17][18][19] . This low expression rate of PD-L1 may be attributed to correlated factors, including a patient cohort, ethnic differences, different types of tumor samples or staging, IHC staining method, and positive cutoff levels for PD-L1 expression. Our study used the IHC 22C3 pharmDx, the only companion diagnostic assay approved by the FDA, at the CPS ≥ 1 and ≥ 5 cutoffs to assess the PD-L1 expression in GC 20 . Moreover, we used surgical resection samples to avoid intratumoral heterogenicity from biopsy specimens and for precise pathological staging. Although, our study found no statistically significant correlation between PD-L1 positivity and gender, pTNM stage, Lauren classification, tumor location, tumor size, lymphatic invasion, vascular invasion, or metastases. However, PD-L1, CPS ≥ 1 and PD-L1, CPS ≥ 5 had a statistically significant correlation with age lower than 55 (32.6% vs. 16.5%, p = 0.003; 11.6% vs. 4.4%, p = 0.027). Consistent with the previous report, PD-L1 expression was more common in young-onset than average-onset GC patients (31% vs. 3%, p < 0.05) 21 .
GC is an epithelial tumor associated with Epstein-Barr virus (EBV) infection confirmed by EBV type A and wild-type LMP1 variants in GC lesions in the Thai population 22 . Based on epidemiological data, 95% of adult Thais have immunity to EBV from childhood infection 23 . Thus, EBV-positive GC is found in younger patients more often than in EBV-negative gastric tumors 24 . More than half of the GC patients in our study have been infected with EBV. Likewise, in the previous studies from Brazil and Turkey, the positivity of EBV was 50% to 60% in gastric cancer tissues 25,26 . EBV induces intra-or peri-tumoral immune cell infiltration and shows genomic amplification of the chromosome 9 locus containing the genes encoding PD-L1 27 . In addition, EBV has upregulated expression levels of PD-L1 in cancer and immune cells 28 . Consequently, overexpression of PD-L1 is observed in young patients with EBV-associated GC 29,30 . Moreover, elderly patients have low levels of PD-L1 expression due to immune senescence caused by thymic involution and decreased synthesis of T cell progenitors from bone marrow 31 . These reasons explain the results of our study showing that PD-L1 positivity was more common in young Thai patients than in elderly patients with GC. We hypothesize that EBV plays a role in the pathogenesis of GC by enhancing PD-L1 expression and provides potentially relevant biomarkers for selecting patients who may derive more significant benefits from PD-1/PD-L1 checkpoint inhibitors, an emerging novel treatment option for GC.
The impact of PD-L1 expression on prognosis remains controversial in several malignancies 19,[32][33][34][35] . In our study, PD-L1 positivity in Thai patients with GC was associated with poor prognosis and higher mortality, reducing the chances of overall survival. These findings are related to the PD-L1 positivity, which was more common in patients with metastases than without. Supporting our findings, a meta-analysis on GC patients revealed that PD-L1 positivity corresponded to a poor prognosis for overall survival 36,37 . Patients with PD-L1 expression should receive immunotherapy instead of standard-of-care for GC. Therefore, PD-L1 expression can be used as a reliable indicator for monitoring the clinical prognosis of GC patients.
However, there are certain limitations of this study. This study was a retrospective analysis that used archived tissue specimens from tissue blocks which likely influenced the amount of PD-L1 expression that may change over time. In addition, since this was a single-center study, selection bias may have existed. Given these limitations, it is probably improper to consider our results as a wholly accurate representation of the prevalence of PD-L1 expression in GC. A well-conducted prospective randomized multicenter trial can give us the exact prevalence of PD-L1 expression and its clinicopathological correlation with GC in Thailand. However, our study can provide insights for improving the selection of patients eligible for anti-PD-1/PD-L1 therapy.

Conclusion
Accurate assessment of PD-L1 expression in GC in the Thai population provides valuable data unique to Thai patients and allows for the cost-effective management of cancer in this population. PD-L1 expression was evident in one-fourth of Thai patients with GC. Furthermore, the expression of PD-L1 has been associated with young age, short survival, and promoting metastases, although unrelated to the tumor stage. Therefore, PD-L1 testing is recommended, especially among young GC patients with metastases, to select patients eligible for anti-PD-1/ PD-L1 therapy.

Data availability
Datasets analyzed during the current study are available from the corresponding author upon reasonable request.